Hypoxic Pulmonary Hypertension In Mice Research Articles

EPAS1 (Endothelial PAS Domain Protein 1) Orchestrates Transactivation of Endothelial ICAM1 (Intercellular Adhesion Molecule 1) by Small Nucleolar RNA Host Gene 5 (SNHG5) to Promote Hypoxic Pulmonary Hypertension.

[Figure: see text].

Induction of Mucosal Tolerance to Collagen Type V Attenuates Hypoxic Pulmonary Hypertension

Hypoxic pulmonary hypertension (PH) can be caused by lung disease, injury, and chronic hypoxia (CH) exposure. Hallmarks for PH include recruitment of immune cells to the perivascular region of the lungs, thickening of the pulmonary arterial walls, elevated pulmonary vascular resistance and pressure, and right ventricular remodeling. These signature signs of PH are what lead to right heart failure and ultimately death. We have previously reported that 21 days of CH leads to both an upregulation of collagen type V (Col V) as well as natural T helper 17 (nTh17) cell-reactivity towards Col V in mouse pulmonary arteries without affecting the number of T regulatory cells (Tregs) in the lungs. However, it is unknown if Treg suppressive capacity and/or peripheral tolerance to autoantigens is affected by CH. Col V-specific Tregs, e.g. peripheral tolerance, can be induced by nasal instillation of Col V without adjuvant. Using right ventricular systolic pressure (RVSP) and pulmonary arterial wall thickening as indicators of PH, we hypothesized that induction of mucosal tolerance to Col V attenuates hypoxic PH in mice. Our data support this hypothesis, as both RVSP and wall thickening are lower in CH-exposed Col V-inoculated vs. vehicle-inoculated mice (Figure). Future studies will characterize the effect of CH on Treg suppressive activity, nTh17 perivascular infiltration, and nTh17-mediated Col V reactivity. In conclusion, our study suggests that induction of mucosal tolerance to Col V attenuates chronic hypoxia-induced PH.

The effects of genetic deletion of Macrophage migration inhibitory factor on the chronically hypoxic pulmonary circulation.

While it is well established that the haemodynamic cause of hypoxic pulmonary hypertension is increased pulmonary vascular resistance, the molecular pathogenesis of the increased resistance remains incompletely understood. Macrophage migration inhibitory factor is a pleiotropic cytokine with endogenous tautomerase enzymatic activity as well as both intracellular and extracellular signalling functions. In several diseases, macrophage migration inhibitory factor has pro-inflammatory roles that are dependent upon signalling through the cell surface receptors CD74, CXCR2 and CXCR4. Macrophage migration inhibitory factor expression is increased in animal models of hypoxic pulmonary hypertension and macrophage migration inhibitory factor tautomerase inhibitors, which block some of the functions of macrophage migration inhibitory factor, and have been shown to attenuate hypoxic pulmonary hypertension in mice and monocrotaline-induced pulmonary hypertension in rats. However, because of the multiple pathways through which it acts, the integrated actions of macrophage migration inhibitory factor during the development of hypoxic pulmonary hypertension were unclear. We report here that isolated lungs from adult macrophage migration inhibitory factor knockout (MIF–/–) mice maintained in normoxic conditions showed greater acute hypoxic vasoconstriction than the lungs of wild type mice (MIF+/+). Following exposure to hypoxia for three weeks, isolated lungs from MIF–/– mice had significantly higher pulmonary vascular resistance than those from MIF+/+ mice. The major mechanism underlying the greater increase in pulmonary vascular resistance in the hypoxic MIF–/– mice was reduction of the pulmonary vascular bed due to an impairment of the normal hypoxia-induced expansion of the alveolar capillary network. Taken together, these results demonstrate that macrophage migration inhibitory factor plays a central role in the development of the pulmonary vascular responses to chronic alveolar hypoxia.

Consequence of Desmin gene mutation on development of chronic hypoxic pulmonary hypertension in mice

Desmin deficiency is responsible for various forms of skeletal and cardiac disorders. However, role of desmin in the normal and pathological pulmonary vasculature remains unknown. To investigate whether desmin deficiency affects pulmonary circulation in normoxic and hypoxic conditions. Desmin-deficient mice (Des-/-) and their littermate Des+/+ (controls) aged 2–4 months were exposed to chronic hypoxia (9% O2, 2 weeks) or normoxia. Cardiac function was assessed by closed-chest echocardiography and right ventricle (RV) hemodynamics combined with measurements of Fulton Index and muscularization of pulmonary arteries (PA). Contractile properties of intrapulmonary arteries were measured by using an arteriography. Heart and lung phenotype were determined by using standard techniques of histology, immunostaining, immunoblot and RT-qPCR analysis. Under normal conditions Des-/- mice developed cardiomyopathy with alteration of both left ventricle (LV) and RV functions. Left Heart Disease was characterized by ventricular dilatation, decreased Ejection Fraction and Strain Rate in agreement with extreme transmural fibrosis and cardiomyocytes hypertrophy. RV dysfunction was evidenced by an increase of RV systolic pressure (RVSP) and PA muscularization. After exposure to hypoxia, LV function was globally not modified in both Des-/- and Des+/+ mice. RV was similarly affected in both strains, though the increase of RV weight, RVSP and PA muscularization were higher in Des-/- mice. Conversely, contractile capacities of PA were not modified in Des-/- mice in normoxia. Under hypoxia, we recorded modifications of several contractile, structural and Ca2 ± handling proteins in PA - smooth muscle cells (SMC). Our results indicate that desmin can be replaced by vimentin in SMC without loss of function. However, clinical features of desminopathies can also include pulmonary hypertension with PA remodeling and higher RV dysfunction.

Alpha-enolase regulates the malignant phenotype of pulmonary artery smooth muscle cells via the AMPK-Akt pathway

The molecular mechanisms underlying the metabolic shift toward increased glycolysis observed in pulmonary artery smooth muscle cells (PASMC) during the pathogenesis of pulmonary arterial hypertension (PAH) are not fully understood. Here we show that the glycolytic enzyme α-enolase (ENO1) regulates the metabolic reprogramming and malignant phenotype of PASMC. We show that ENO1 levels are elevated in patients with associated PAH and in animal models of hypoxic pulmonary hypertension (HPH). The silencing or inhibition of ENO1 decreases PASMC proliferation and de-differentiation, and induces PASMC apoptosis, whereas the overexpression of ENO1 promotes a synthetic, de- differentiated, and apoptotic-resistant phenotype via the AMPK-Akt pathway. The suppression of ENO1 prevents the hypoxia-induced metabolic shift from mitochondrial respiration to glycolysis in PASMC. Finally, we find that pharmacological inhibition of ENO1 reverses HPH in mice and rats, suggesting ENO1 as a regulator of pathogenic metabolic reprogramming in HPH.

Protective Role of Myelocytic Nitric Oxide Synthases against Hypoxic Pulmonary Hypertension in Mice.

Nitric oxide (NO), synthesized by NOSs (NO synthases), plays a role in the development of pulmonary hypertension (PH). However, the role of NO/NOSs in bone marrow (BM) cells in PH remains elusive. To determine the role of NOSs in BM cells in PH. Experiments were performed on 36 patients with idiopathic pulmonary fibrosis and on wild-type (WT), nNOS (neuronal NOS)-/-, iNOS (inducible NOS)-/-, eNOS (endothelial NOS)-/-, and n/i/eNOSs-/- mice. In the patients, there was a significant correlation between higher pulmonary artery systolic pressure and lower nitrite plus nitrate levels in the BAL fluid. In the mice, hypoxia-induced PH deteriorated significantly in the n/i/eNOSs-/- genotype and, to a lesser extent, in the eNOS-/- genotype as compared with the WT genotype. In the n/i/eNOSs-/- genotype exposed to hypoxia, the number of circulating BM-derived vascular smooth muscle progenitor cells was significantly larger, and transplantation of green fluorescent protein-transgenic BM cells revealed the contribution of BM cells to pulmonary vascular remodeling. Importantly, n/i/eNOSs-/--BM transplantation significantly aggravated hypoxia-induced PH in the WT genotype, and WT-BM transplantation significantly ameliorated hypoxia-induced PH in the n/i/eNOSs-/- genotype. A total of 69 and 49 mRNAs related to immunity and inflammation, respectively, were significantly upregulated in the lungs of WT genotype mice transplanted with n/i/eNOSs-/--BM compared with those with WT-BM, suggesting the involvement of immune and inflammatory mechanisms in the exacerbation of hypoxia-induced PH caused by n/i/eNOSs-/--BM transplantation. These results demonstrate that myelocytic n/i/eNOSs play an important protective role in the pathogenesis of PH.

Role of Ryanodine Receptor Channel and Mechanisms of Vascular Diseases

The functional importance of ryanodine receptors (RyRs) is well established in cardiac and skeletal diseases, but not in smooth muscle diseases. Here we explored the potentially essential role of RyRs in pulmonary hypertension. Using real-time RT-PCR and Western blot analysis, we found that all three RyR subtypes (RyR1, RyR2 and RyR3) were expressed in pulmonary artery smooth muscle cells (PASMCs). Studies with gene knockout (KO) mice revealed that RyR1, RyR2 and RyR3 all showed their functional activity. Contractile and calcium responses in PASMCs following acute hypoxia for 5 and 30 min respectively were completely blocked in RyR2 KO mice, but only partially inhibited by RyR1 or RyR3 KO mice. RyR channel activity and calcium release were largely increased in PASMCs from mice with pulmonary hypertension induced by chronic hypoxia for 21 days. The chronic hypoxia-induced increases in RyR channel activity and calcium release are fully abolished in PASMCs from SMC-specific RyR2 KO mice. Pulmonary artery vasoconstriction, remodeling, and hypertension following chronic hypoxia are all nulled in RyR2 KO mice. FK506 binding protein 12.6 (FKBP12.6, the RyR2 channel inhibitor) is significantly dissociated from the channel in PASMCs from mice with hypoxia-induced pulmonary hypertension. FKBP12.6 KO promoted, while FKBP12.6 overexpression diminished, hypoxic pulmonary hypertension in mice. Treatment of S107, a specific RyR2/FKBP12.6 stabilizer, also protects against hypoxic pulmonary hypertension. Rieske iron-sulfur protein (RISP) in mitochondrial complex III serves as an essential, primary molecule in hypoxia-evoked reactive oxygen species (ROS) generation and subsequent FKBP12.6/RyR2 dissociation in PASMCs. Intravenous administration of lentiviral RISP shRNAs eliminates hypoxia-induced pulmonary hypertension in mice. Collectively, we conclude that hypoxia can cause RISP-mediated ROS production, FKBP12.6/RyR2 dissociation, RyR2 channel hyperfunction, sarcoplasmic reticulum calcium leaking, increased calcium signaling, PASMC proliferation and remodeling, and ultimately pulmonary hypertension.

Protective Role of Myelocytic Nitric Oxide Synthases in Hypoxic Pulmonary Hypertension in Mice

Protective Role of Myelocytic Nitric Oxide Synthases in Hypoxic Pulmonary Hypertension in Mice

Extracellular Calpain/Calpastatin Balance Is Involved in the Progression of Pulmonary Hypertension.

Excessive growth of pulmonary arterial (PA) smooth muscle cells (SMCs) is a major component of PA hypertension (PAH). The calcium-activated neutral cysteine proteases calpains 1 and 2, expressed by PASMCs, contribute to PH but are tightly controlled by a single specific inhibitor, calpastatin. Our objective was to investigate calpastatin during pulmonary hypertension (PH) progression and its potential role as an intracellular and/or extracellular effector. We assessed calpains and calpastatin in patients with idiopathic PAH and mice with hypoxic or spontaneous (SM22-5HTT(+) strain) PH. To assess intracellular and extracellular roles for calpastatin, we studied effects of the calpain inhibitor PD150606 on hypoxic PH in mice with calpastatin overexpression driven by the cytomegalovirus promoter (CMV-Cast) or C-reactive protein (CRP) promoter (CRP-Cast), inducing increased calpastatin production ubiquitously and in the liver, respectively. Chronically hypoxic and SM22-5HTT(+) mice exhibited increased lung calpastatin and calpain 1 and 2 protein levels and activity, both intracellularly and extracellularly. Prominent calpastatin and calpain immunostaining was found in PASMCs of remodeled vessels in mice and patients with PAH, who also exhibited increased plasma calpastatin levels. CMV-Cast and CRP-Cast mice showed similarly decreased PH severity compared with wild-type mice, with no additional effect of PD150606 treatment. In cultured PASMCs from wild-type and CMV-Cast mice, exogenous calpastatin decreased cell proliferation and migration with similar potency as PD150606 and suppressed fibronectin-induced potentiation. These results indicate that calpastatin limits PH severity via extracellular mechanisms. They suggest a new approach to the development of treatments for PH.

Effects of gender on severity and pulmonary artery vascular reactivity in chronic hypoxic pulmonary hypertension in mice

To explore the effects of gender on pathology and pulmonary vascular reactivity in chronic hypoxic pulmonary hypertension (HPH) in mice. HPH was induced by a 4-week exposure of male and female mice (8 weeks old C57BL/6, n = 15 each) to chronic hypoxia (10%O2). Normoxic controls were established both in male and female groups (n = 15 each). Right ventricular systolic pressure (RVSP) and Fulton index were detected.Isolated pulmonary artery ring contraction induced by high potassium (K(+)) and U-46619 was recorded and compared between two groups. Chronic hypoxia induced pulmonary hypertension after 4 weeks.RVSP significantly increased both in hypoxic female and male groups (normoxic vs hypoxic: (23.40 ± 0.16) vs (31.46 ± 2.33) and (24.71 ± 0.44) vs (33.66 ± 1.38) mmHg (1 mmHg = 0.133 kPa), P = 0.029 and P < 0.001).However, there were no inter-group differences (P = 0.447). The Fulton index of hypoxic female and male mice was higher than normoxic counterparts (P = 0.001, P < 0.001). But there were no gender differences (P = 0.471). Contraction of high K(+) and U-46619 was calcium ion (Ca(2+))-dependent and decreased markedly without calcium (P < 0.001, P = 0.007). High K(+) and U-46619-induced contraction of pulmonary artery was concentration dependent and greater in chronic hypoxic female (P = 0.002, P < 0.001) and male mice (both P < 0.001). But no differences existed between male and female mice in high K(+) (P = 0.657) or U-46619 (P = 0.751)-induced contraction. No significant correlation exists between gender and occurrence and severity of pulmonary hypertension or pulmonary artery vascular reactivity in HPH mice.

A Sex-Specific MicroRNA-96/5-Hydroxytryptamine 1B Axis Influences Development of Pulmonary Hypertension.

Females are predisposed to pulmonary arterial hypertension (PAH); evidence suggests that serotonin, mutations in the bone morphogenetic protein receptor (BMPR) II gene, and estrogens influence development of PAH. The 5-hydroxytryptamine 1B receptor (5-HT1BR) mediates human pulmonary artery smooth muscle cell (hPASMC) proliferation. We aimed to determine whether selected microRNAs (miRNAs) expressed in PASMCs are influenced by sex, BMPR-II mutations, and estrogens, and contribute to PASMC proliferation in PAH. Expression levels of miRNAs targeting genes related to PAH, estrogen, and serotonin were determined by quantitative RT-PCR in hPASMCs and mouse PASMCs harboring a heterozygous mutation in BMPR-II (BMPR-II(R899X+/-) PASMCs). miRNA-96 targets 5-HT1BR and was selected for further investigation. miRNA target validation was confirmed by luciferase reporter assay. Precursor miRNA-96 was transfected into hPASMCs to examine effects on proliferation and 5-HT1BR expression. The effect of a miRNA-96 mimic on the development of hypoxic pulmonary hypertension in mice was also assessed. miRNA-96 expression was reduced in BMPR-II(R899X+/-) PASMCs from female mice and hPASMCs from female patients with PAH; this was associated with increased 5-HT1BR expression and serotonin-mediated proliferation. 5-HT1BR was validated as a target for miRNA-96. Transfection of precursor miRNA-96 into hPASMCs reduced 5-HT1BR expression and inhibited serotonin-induced proliferation. Restoration of miRNA-96 expression in pulmonary arteries in vivo via administration of an miRNA-96 mimic reduced the development of hypoxia-induced pulmonary hypertension in the mouse. Increased 5-HT1BR expression may be a consequence of decreased miRNA-96 expression in female patient PASMCs, and this may contribute to the development of PAH.

Elafin Reverses Pulmonary Hypertension via Caveolin-1-Dependent Bone Morphogenetic Protein Signaling.

Pulmonary arterial hypertension is characterized by endothelial dysfunction, impaired bone morphogenetic protein receptor 2 (BMPR2) signaling, and increased elastase activity. Synthetic elastase inhibitors reverse experimental pulmonary hypertension but cause hepatotoxicity in clinical studies. The endogenous elastase inhibitor elafin attenuates hypoxic pulmonary hypertension in mice, but its potential to improve endothelial function and BMPR2 signaling, and to reverse severe experimental pulmonary hypertension or vascular pathology in the human disease was unknown. To assess elafin-mediated regression of pulmonary vascular pathology in rats and in lung explants from patients with pulmonary hypertension. To determine if elafin amplifies BMPR2 signaling in pulmonary artery endothelial cells and to elucidate the underlying mechanism. Rats with pulmonary hypertension induced by vascular endothelial growth factor receptor blockade and hypoxia (Sugen/hypoxia) as well as lung organ cultures from patients with pulmonary hypertension were used to assess elafin-mediated reversibility of pulmonary vascular disease. Pulmonary arterial endothelial cells from patients and control subjects were used to determine the efficacy and mechanism of elafin-mediated BMPR2 signaling. In Sugen/hypoxia rats, elafin reduced elastase activity and reversed pulmonary hypertension, judged by regression of right ventricular systolic pressure and hypertrophy and pulmonary artery occlusive changes. Elafin improved endothelial function by increasing apelin, a BMPR2 target. Elafin induced apoptosis in human pulmonary arterial smooth muscle cells and decreased neointimal lesions in lung organ culture. In normal and patient pulmonary artery endothelial cells, elafin promoted angiogenesis by increasing pSMAD-dependent and -independent BMPR2 signaling. This was linked mechanistically to augmented interaction of BMPR2 with caveolin-1 via elafin-mediated stabilization of endothelial surface caveolin-1. Elafin reverses obliterative changes in pulmonary arteries via elastase inhibition and caveolin-1-dependent amplification of BMPR2 signaling.

Differential effects of CX3CR1 and CCR2 deficiencies in hypoxic pulmonary hypertension

Introduction The development of hypoxia-induced pulmonary hypertension (PH) is associated with lung inflammation and vessel infiltration by monocytes. Two major monocyte subsets showing differential expression of CCR2 and CX3CR1 receptors have been identified. Ly6C hi monocytes are involved in inflammation and express high levels of CCR2 but low levels of CX3CR1 (Ly6C hi CCR2 hi /CX3CR1 lo ). Ly6C lo monocytes are the noninflammatory subtype and express high levels of CX3CR1 (Ly6C l °CCR2 lo /CX3CR1 hi ). Methods To determine the roles for Ly6C hi and Ly6C lo monocytes in hypoxic PH in mice, we studied CX3CR1 -/- , CCL2 -/− or double CX3CR1/CCL2 -/− mice compared to wild type (WT) mice. Results Development of hypoxia-induced PH was associated with a marked increase in lung mRNA levels of CX3CR1, CCR2 and of their respective ligands CX3CL1 and CCL2. FACs analysis revealed sustained increases in both monocyte subsets in blood but only a transient peak on day 3 of hypoxia exposure in lung tissue. Total lung macrophage counts remained unchanged. CX3CR1 -/− mice showed protection against hypoxic PH compared to WT mice, whereas CCL2 -/− mice and double CX3CR1/CCL2 -/− mice exhibited similar PH severity as did WT mice. Chronically hypoxic WT and mutant mice did not differ regarding lung Ly6C lo monocytes, whereas Ly6C hi counts were reduced in CCL2 -/− or double CX3CR1/CCL2 -/− mice compared to WT or CX3CR1 -/− mice. Total lung macrophage counts did not differ between WT and CCL2 -/− or double CX3CR1/CCL2 -/− mice but were markedly increased in CX3CR1 -/− mice. These results are consistent with previous studies showing that CCR2 deficiency exacerbates PH in hypoxic mice, and they support a prominent effect of the CX3CL1/CX3CR1 axis on pulmonary vascular remodeling. Accordingly, we found that CX3CL1 potentiated proliferation of cultured PA-SMCs from WT mice but not from CX3CR1 -/− mice. Conclusion CX3CR1 deficiency protects against hypoxia-induced PH by modulating monocyte recruitment and PA-SMC cell proliferation.

Abstract 13284: A Novel Formulation of Vasoactive Intestinal Peptide Attenuates both Acute Hypoxic Pulmonary Vasoconstriction and the Development of Pulmonary Hypertension

Background: Vasoactive intestinal peptide (VIP) is an endogenous hormone that is known to relax vascular smooth muscle and has established anti-proliferative and immunomodulatory effects in the pulmonary circulation making it an attractive therapeutic target in pulmonary arterial hypertension (PAH). In the current study, a polymer-based nanocarrier (protected graft copolymer - PGC) formulation of VIP, which has been shown to increase the potency and duration of action of VIP, is used to show both acute vasodilatory effects and chronic therapeutic effects in experimental animal models of pulmonary hypertension. Methods: The isolated perfused mouse lung preparation is utilized to test acute hypoxic pulmonary vasoconstriction (HPV) in mice. Two animal models of pulmonary hypertension are used in preventative experiments, chronic hypoxic pulmonary hypertension in mice and monocrotaline-induced pulmonary hypertension in rats. Right ventricular systolic pressure and Fulton’s index (weight ratio of RV/[LV+Septum]) are used for measures of pulmonary hemodynamics and RV hypertrophy respectively. Results: PGC-VIP decreased resting pulmonary artery pressure and attenuated acute HPV elicited by 1% inhaled oxygen tension in a dose dependent manner from 0.1 μM to 1.0 μM. After four weeks of chronic hypoxia, both RVSP measurements and Fulton’s index were significantly decreased in mice receiving 100 mg/kg intraperitoneal PGC-VIP every other day compared to vehicle control. Higher doses were associated with mortality in the treatment group. MCT-PH rats receiving subcutaneous PGC-VIP at a dose of 250 mg/kg failed to show improvement in RVSP or Fulton’s index compared to vehicle control. Conclusion: This novel formulation of VIP demonstrates both acute and chronic vasodilatory effects in the pulmonary circulation. Treatment with PGC-VIP can attenuate the development of hypoxic pulmonary hypertension, yet significant mortality is seen at higher doses. Subcutaneous injection failed to attenuate the development of experimental PH in rats, possibly due to an ineffective dose or route of administration. Further studies are underway to identify the ideal dosing strategy necessary to attenuate and potentially reverse experimental PH in animal models.

Abstract 316: Angiotensin 1-7 Regulation Of Endothelin-1 System In Pulmonary Arterial Hypertension

Studies suggest that activation of ACE2-Ang-(1-7)-Mas ameliorates vascular, cardiac and lung damage observed in pulmonary hypertension (PAH); a condition where endothelin-1 (ET-1) is important. Here we assessed whether Ang 1-7 regulates ET-1 system in pulmonary hypertension and putative mechanisms. Hypobaric hypoxia was used to induce hypoxic PAH in mice, which were divided in 4 groups: normoxic controls (NC), hypoxic PAH (HP), normoxic (NA) and hypoxic PAH (HA) treated with Ang 1-7 (hydroxypropyl-βcyclodextrin-Ang(1-7) 30μg/kg/day given by oral gavage for 14 days after established hypoxia-induced PAH. In HP mice, RVSP (18.7 NC vs. 47.6mmHg HP, p&lt;0.001), RVH by Fulton Index (0.198 NC vs. 0.279 HP, p&lt;0.01) and urinary ET-1 levels (0.88 NC vs 2.48pg/ml HP, p&lt;0.05 vs NC) were increased, an effect blocked by Ang1-7 treatment. In NA, Ang 1-7 increased urinary levels of ET-1 (2.24pg/ml, p&lt;0.05 vs NC). No changes were observed on ETBR protein expression between all groups. Human microvascular endothelial cells (HMEC) were also used and stimulated with ET-1 in the absence/presence of Ang 1-7. Ang 1-7 stimulation increased preproET-1 mRNA levels (250%), ET-1 release (125%), and ETBR protein levels (50%), p&lt;0.05 vs vehicle. To examine whether the regulation of ET-1 system by Ang 1-7 is beneficial, HMEC were stimulated with ET-1 in the absence/presence of Ang 1-7. ET-1 increased e-selectin mRNA (400%) and VCAM-1 protein (38%) levels, as well as TNFα production (30%); all effects blocked by Ang 1-7 (p&lt;0.05). Ang 1-7 inhibition of ET-1 pro-inflammatory effects was dependent on NO production, since it was blocked by L-NAME and LY294002 (inhibitors of NO pathway). Interestingly, Ang 1-7 increased NO production (257%) through Mas and ETBR-dependent manner. An interaction between Mas and ETbR was observed in HMEC by immunoprecipitation and peptide array protocols. In conclusion, Ang 1-7 modulates ET-1 system in pulmonary hypertension and HMEC. Our findings indicate that Ang 1-7 negatively modulates proinflammatory signalling in human endothelial cells. The protective effect may involve upregulation/crosstalk of/with ETBR. These data identify the ET-1 system as a novel molecular mechanism involved in the putative protective action of Ang 1-7 in pulmonary hypertension.

Prevention of hypoxic pulmonary hypertension by hypoxia-inducible expression of p27 in pulmonary artery smooth muscle cells.

Hypoxia-induced proliferation of pulmonary artery smooth muscle cells (SMCs) is important in the development of hypoxic pulmonary hypertension (HPH). We constructed a lentivirial vector containing a smooth muscle-specific promoter and six copies of hypoxia response element to co-drive the expression of p27, the key cyclin-dependent kinase inhibitor that blocks the G1 to S phase transition in cell cycle progression, in pulmonary artery SMCs in hypoxia. Then in vivo we examined the prevention effects of the vector on HPH in mice and in vitro the specificity on the hypoxia-inducible expression of p27 in pulmonary artery SMCs. Hypobaric hypoxia for 4 weeks resulted in significant increases in the right ventricular systolic pressure, the ratio of right ventricle to left ventricle plus septal weight and the muscularization of pulmonary vessels in mice. Administration of the vector before hypoxia significantly prevented the effects of hypoxia. In vitro, the vector exhibited hypoxic inducibility and relatively specific expression in pulmonary artery SMCs, inhibited the hypoxia-induced proliferation of pulmonary artery SMCs and arrested more cells at G0/G1 phase. These results demonstrate that the hypoxia-inducible p27 expression prevents the development of HPH in mice.

Deletion of STAT5a/b in vascular smooth muscle abrogates the male bias in hypoxic pulmonary hypertension in mice: implications in the human disease.

Chronic hypoxia typically elicits pulmonary hypertension (PH) in mice with a male-dominant phenotype. There is an opposite-sex bias in human PH, with a higher prevalence in women, but greater survival (the "estrogen paradox"). We investigated the involvement of the STAT5a/b species, previously established to mediate sexual dimorphism in other contexts, in the sex bias in PH. Mice with heterozygous or homozygous deletions of the STAT5a/b locus in vascular smooth muscle cells (SMCs) were generated in crosses between STAT5a/b(fl/fl) and transgelin (SM22α)-Cre(+/+) parents. Wild-type (wt) males subjected to chronic hypoxia showed significant PH and pulmonary arterial remodeling, with wt females showing minimal changes (a male-dominant phenotype). However, in conditional STAT5(+/-) or STAT5(-/-) mice, hypoxic females showed the severest manifestations of PH (a female-dominant phenotype). Immunofluorescence studies on human lung sections showed that obliterative pulmonary arterial lesions in patients with idiopathic pulmonary arterial hypertension (IPAH) or hereditary pulmonary arterial hypertension (HPAH), both male and female, overall had reduced STAT5a/b, reduced PY-STAT5 and reduced endoplasmic reticulum (ER) GTPase atlastin-3 (ATL3). Studies of SMCs and endothelial cell (EC) lines derived from vessels isolated from lungs of male and female IPAH patients and controls revealed instances of coordinate reductions in STAT5a, STAT5b and ATL3 in IPAH-derived cells, including SMCs and ECs from the same patient. Taken together, these data provide the first definitive evidence for a contribution of STAT5a/b to the sex bias in PH in the hypoxic mouse and implicate reduced STAT5 in the pathogenesis of the human disease.

Biopterin Metabolism and eNOS Expression during Hypoxic Pulmonary Hypertension in Mice

Tetrahydrobiopterin (BH4), which fosters the formation of and stabilizes endothelial NO synthase (eNOS) as an active dimer, tightly regulates eNOS coupling / uncoupling. Moreover, studies conducted in genetically-modified models demonstrate that BH4 pulmonary deficiency is a key determinant in the pathogenesis of pulmonary hypertension. The present study thus investigates biopterin metabolism and eNOS expression, as well as the effect of sepiapterin (a precursor of BH4) and eNOS gene deletion, in a mice model of hypoxic pulmonary hypertension. In lungs, chronic hypoxia increased BH4 levels and eNOS expression, without modifying dihydrobiopterin (BH2, the oxidation product of BH4) levels, GTP cyclohydrolase-1 or dihydrofolate reductase expression (two key enzymes regulating BH4 availability). In intrapulmonary arteries, chronic hypoxia also increased expression of eNOS, but did not induce destabilisation of eNOS dimers into monomers. In hypoxic mice, sepiapterin prevented increase in right ventricular systolic pressure and right ventricular hypertrophy, whereas it modified neither remodelling nor alteration in vasomotor responses (hyper-responsiveness to phenylephrine, decrease in endothelium-dependent relaxation to acetylcholine) in intrapulmonary arteries. Finally, deletion of eNOS gene partially prevented hypoxia-induced increase in right ventricular systolic pressure, right ventricular hypertrophy and remodelling of intrapulmonary arteries. Collectively, these data demonstrate the absence of BH4/BH2 changes and eNOS dimer destabilisation, which may induce eNOS uncoupling during hypoxia-induced pulmonary hypertension. Thus, even though eNOS gene deletion and sepiapterin treatment exert protective effects on hypoxia-induced pulmonary vascular remodelling, increase on right ventricular pressure and / or right ventricular hypertrophy, these effects appear unrelated to biopterin-dependent eNOS uncoupling within pulmonary vasculature of hypoxic wild-type mice.

Digoxin inhibits development of hypoxic pulmonary hypertension in mice

Chronic hypoxia is an inciting factor for the development of pulmonary arterial hypertension. The mechanisms involved in the development of hypoxic pulmonary hypertension (HPH) include hypoxia-inducible factor 1 (HIF-1)-dependent transactivation of genes controlling pulmonary arterial smooth muscle cell (PASMC) intracellular calcium concentration ([Ca(2+)](i)) and pH. Recently, digoxin was shown to inhibit HIF-1 transcriptional activity. In this study, we tested the hypothesis that digoxin could prevent and reverse the development of HPH. Mice were injected daily with saline or digoxin and exposed to room air or ambient hypoxia for 3 wk. Treatment with digoxin attenuated the development of right ventricle (RV) hypertrophy and prevented the pulmonary vascular remodeling and increases in PASMC [Ca(2+)](i), pH, and RV pressure that occur in mice exposed to chronic hypoxia. When started after pulmonary hypertension was established, digoxin attenuated the hypoxia-induced increases in RV pressure and PASMC pH and [Ca(2+)](i). These preclinical data support a role for HIF-1 inhibitors in the treatment of HPH.

Hypoxic Pulmonary Hypertension in Mice with Constitutively Active Platelet‐Derived Growth Factor Receptor‐β

Platelet-derived growth factor (PDGF) has been implicated in the pathobiology of vascular remodeling. The multikinase inhibitor imatinib that targets PDGF receptor (PDGFR), c-kit and Abl kinases, shows therapeutic efficacy against experimental pulmonary hypertension (PH); however, the role of PDGFR-b in experimental PH has not been examined by genetic approach. We investigated the chronic hypoxia-induced PH in mice carrying an activating point mutation of PDGFR-β (D849N) and evaluated the therapeutic efficacy of imatinib. In addition, we studied pulmonary global gene expression and confirmed the expression of identified genes by immunohistochemistry. Chronically hypoxic D849N mice developed PH and strong pulmonary vascular remodeling that was improved by imatinib (100 mg/kg/day) as evident from the significantly reduced right ventricular systolic pressure, right ventricular hypertrophy and muscularization of peripheral pulmonary arteries. Global gene expression analysis revealed that stromal cell derived factor SDF)-1α was significantly upregulated, which was confirmed by immunohistochemistry. Moreover, an enhanced immunoreactivity for SDF-1α, PDGFR-β and CXCR4, the receptor for SDF-1α was localized to the α-smooth muscle cell (SMC) actin positive pulmonary vascular cells in hypoxic mice and patients with idiopathic pulmonary arterial hypertension (IPAH). In conclusion, our findings substantiate the major role of PDGFR activation in pulmonary vascular remodeling by a genetic approach. Immunohistochemistry findings suggest a role for SDF-1α/CXCR4 axis in pulmonary vascular remodeling and point to a potential interaction between the chemokine SDF-1 and the growth factor PDGF signaling. Future studies designed to elucidate an interaction between the chemokine SDF-1 and the PDGF system may uncover novel therapeutic targets.